Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy. A key factor contributing to the lethality of PDAC is immune evasion. Tumor cells suppress anti-tumor immunity by activating immune checkpoint proteins on immune cells including macrophages. Siglec receptors are one of the main checkpoint molecules on macrophages. The ligand for Siglecs is sialic acid. To effectuate immune suppression via Siglecs, PDAC cells increase their surface sialylation by upregulating the expression of sialyltransferases such as ST6GAL1. ST6GAL1 adds an α2,6-linked sialic acid to N-glycans. Both α2,6 sialylation and ST6GAL1 expression are markedly increased in PDAC cells. Our prior studies revealed potent tumor-autonomous functions for ST6GAL1. Our new unpublished results suggest that the tumorigenic effects of ST6GAL1 are also driven by its role in creating sialoglycan ligands for macrophage Siglecs. Macrophages that are polarized to an immunosuppressive M2 phenotype are key contributors to PDAC progression. The goal of our study is to determine whether macrophage Siglecs are promising therapeutic targets for preventing M2 polarization, thus restoring anti-tumor immunity and preventing PDAC progression. We investigated macrophage polarization in our genetically engineered mouse models of PDAC, utilizing either pancreas-specific knock-in of oncogenic K-ras (KC mouse) or K-ras in combination with ST6GAL1 knock-in (KSC mouse). Single-cell RNA sequencing, flow cytometry, and immunohistochemistry showed an increased number of M2 macrophages in KSC vs. KC pancreata. Macrophages co-cultured with ST6GAL1-overexpressed PDAC cells showed increased expression of M2 markers. But adding Siglec-blocking antibodies reversed the ST6GAL1-mediated M2 polarization, indicating that the ST6GAL-mediated M2 polarization of macrophages is mediated through Siglec signaling. Additionally, we determined the phagocytotic capacity of macrophages, which is an established functional readout for anti-tumor behavior of macrophages. Immunofluorescence microscopy and flow cytometry showed that the phagocytotic capability of macrophages was suppressed by ST6GAL1-overexpressed PDAC cells but recovered by Siglec blocking antibody. Overall, our data demonstrate that α2,6 sialic acids on PDAC cells engage with Siglecs on macrophages to induce polarization of macrophages into an immunosuppressive M2 phenotype. Collectively, these results reveal a potential mechanism for targeting Siglecs as a promising immune checkpoint therapy for PDAC patients with high levels of ST6GAL1. Traditional T cell-based checkpoint therapies have limited effectiveness in PDAC. Targeting Siglec checkpoints presents an opportunity to enhance the anti-tumor activity of macrophages, the primary immune cell type within the PDAC tumor microenvironment. Citation Format: Barnita Haldar, Karin Hardiman, Susan Bellis, . Role of sialic acid-siglec axis in pancreatic cancer immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3518.

Glioblastoma (GBM) secreted factors have been identified as key mediators of cellular crosstalk enabling tumor progression. Secreted proteins may be glycosylated, and one form of glycosylation is sialylation. We recently determined that brain tumor initiating cells (BTICs) express elevated levels of the glycosyltransferase beta-galactoside α2,6-sialyltransferase 1 (ST6GAL1). ST6GAL1 adds α2,6 sialic acid to select proteins bound for the cell surface or secreted. In GBM and most cancers, the role of ST6GAL1 in regulating the secretome is completely unknown. Using proteomics screens with BTIC conditioned media, we determined that knockdown of ST6GAL1 altered 1) levels of secreted proteins and 2) levels of α2,6-sialylated proteins. In an unbiased proteomics screen of BTIC conditioned media, levels of seven secreted proteins were decreased and three were increased with two different ST6GAL1 shRNAs in comparison to a non-targeting control. In a separate experiment using a lectin that binds specifically to α2,6-sialic acid, levels of seven proteins were decreased and three were increased. Together, we identified four proteins whose secreted levels and α2,6-sialylation were both ST6GAL1 regulated. Among these, galectin-3-binding protein (LGALS3BP) was of interest because LGALS3BP is upregulated in many cancers, including GBM. LGALS3BP increases tumor growth and progression, including by promoting metastasis through increased cell adhesion, migration, and invasion. We validated by immunoblotting that the secreted form of LGALS3BP was decreased with ST6GAL1 knockdown. Consistent with the known roles of LGALS3BP, treatment of GBM cells with conditioned media from ST6GAL1 knockdown cells altered cell adhesion in comparison to media from non-targeting control cells, although we continue to investigate the impact of ST6GAL1 regulation of LGALS3BP on GBM growth. Overall, this work is the first to determine that α2,6-sialylation by ST6GAL1 is required for maintaining the GBM secretome and that ST6Gal1 plays an important role in the regulation of LGALS3BP secretion and α2,6-sialylation in GBM.

Unlike other post translational modifications like phosphorylation, glycosylation has often been overlooked as a critical regulator of cell signaling in cancers, including brain tumors. One form of glycosylation is sialylation, or the addition of sialic acid to glycoproteins. We recently published that brain tumor initiating cells (BTICs) express high levels of the glycosyltransferase beta-galactoside α2,6-sialytransferase 1 (ST6Gal1). ST6Gal1-mediated α2,6 sialylation in BTICs was critical for glioblastoma growth in vivo. To further identify the molecular targets of ST6Gal1 in BTICs, we performed proteomic profiling of α2,6 sialylated proteins in cell lysates with and without ST6Gal1. When the proteins that were pulled down in non-targeting controls but depleted with ST6Gal1knockdown were evaluated in pathways analysis, there was enrichment for metabolic pathways. Untargeted metabolomics of GBM cells with ST6Gal1 knockdown further confirmed shifts in metabolism with ST6Gal1 loss, notably changes in glycolysis. α2,6 sialylation high GBM cells had increased glucose uptake in comparison to α2,6 sialylation low GBM cells: knockdown of ST6Gal1 with two different shRNAs in BTICs also decreased glucose uptake in comparison to non-targeting control shRNA cells. Knowing that a major regulator of glucose uptake in BTICs is the N-glycoprotein GLUT3, we investigated whether GLUT3 could be a target for ST6Gal1-α2,6 mediated sialylation. Based on its sequence, GLUT3 has a single, potential N-glycan target for α2,6 sialylation. While the biological consequence of GLUT3 α2,6 sialylation remained unknown, ST6Gal1-mediated α2,6 sialylation regulates protein conformation, cell-surface retention, and interactions. Thus, we used high resolution imaging to investigate ST6Gal1 impacts on GLUT3 cell-surface dynamics. We determined that ST6Gal1 knockdown decreased cell surface levels of GLUT3 but did not decrease intracellular levels of GLUT3. Overall, we demonstrate a critical role for ST6Gal1 and α2,6 sialylation in GBM metabolism and define GLUT3 as a novel target of ST6Gal1-mediated α2,6 sialylation.

The TNF-TNFR1 signaling pathway plays a pivotal role in regulating the balance between cell survival and cell death. Upon binding to TNF, plasma membrane-localized TNFR1 initiates survival signaling, whereas TNFR1 internalization promotes caspase-mediated apoptosis. We previously reported that the a 2-6 sialylation of TNFR1 by the tumor-associated sialyltransferase ST6GAL1 diverts signaling toward survival by inhibiting TNFR1 internalization. In the current investigation, we interrogated the mechanisms underlying sialylationdependent regulation of TNFR1 and uncovered a novel role for a 2-6 sialylation, but not a 2-3 sialylation, in mediating apoptosis-resistance. Our studies utilized HEK293 cells with deletion of sialyltransferases that modify N-glycans with either a 2-3-linked sialic acids (ST3GAL3/4/6) or a 2-6-linked sialic acids (ST6GAL1/2). Additionally, ST6GAL1 was re-expressed in cells with ST6GAL1/2 deletion to restore a 2-6 sialylation. Using total internal reflection fl uorescence (TIRF) microscopy and BS3 cross-linking, we determined that, under basal conditions, cells expressing TNFR1 devoid of a 2-6 sialylation displayed enhanced TNFR1 oligomerization, an event that poises cells for activation by TNF. Moreover, upon stimulation with TNF, greater internalization of TNFR1 was observed via time-lapse TIRF and fl ow cytometry, and this correlated with increased caspase-dependent apoptosis. These effects were reversed by ST6GAL1 re-expression. Conversely, eliminating a 2-3 sialylation did not significantly alter TNFR1 clustering, internalization or apoptosis. We also evaluated the Fas receptor, given its structural similarity to TNFR1. As with TNFR1, a 2-6 sialylation had a selective effect in protecting cells against Fas-mediated apoptosis. These results collectively suggest that ST6GAL1 may serve a unique function in shielding cancer cells from apoptotic stimuli within the tumor microenvironment.

3-O-sulfation of heparan sulfate (HS) is the key determinant for binding and activation of antithrombin III (AT). This interaction is the basis of heparin treatment to prevent thrombotic events and excess coagulation. Antithrombin-binding HS (HSAT) is expressed in human tissues but is thought to be expressed in the subendothelial space, mast cells, and follicular fluid. Here, we show that HSAT is ubiquitously expressed in the basement membranes of epithelial cells in multiple tissues. In the pancreas, HSAT is expressed by healthy ductal cells, and its expression is increased in premalignant pancreatic intraepithelial neoplasia lesions but not in pancreatic ductal adenocarcinoma (PDAC). Inactivation of HS3ST1, a key enzyme in HSAT synthesis, in PDAC cells eliminated HSAT expression, induced an inflammatory phenotype, suppressed markers of apoptosis, and increased metastasis in an experimental mouse PDAC model. HSAT-positive PDAC cells bind AT, which inhibits the generation of active thrombin by tissue factor and factor VIIa. Furthermore, plasma from patients with PDAC showed accumulation of HSAT, suggesting its potential as a marker of tumor formation. These findings suggest that HSAT exerts a tumor-suppressing function through recruitment of AT and that the decrease in HSAT during progression of pancreatic tumorigenesis increases inflammation and metastatic potential.

IntroductionColorectal cancer is the third most common cause of cancer death. Rectal cancer makes up a third of all colorectal cases. Treatment for locally advanced rectal cancer includes chemoradiation followed by surgery. We have previously identified ST6GAL1 as a cause of resistance to chemoradiation in vitro and hypothesized that it would be correlated with poor response in human derived models and human tissues.MethodsFive organoid models were created from primary human rectal cancers and ST6GAL1 was knocked down via lentivirus transduction in one model. ST6GAL1 and Cleaved Caspase-3 (CC3) were assessed after chemoradiation via immunostaining. A tissue microarray (TMA) was created from twenty-six patients who underwent chemoradiation and had pre- and post-treatment specimens of rectal adenocarcinoma available at our institution. Immunohistochemistry was performed for ST6GAL1 and percent positive cancer cell staining was assessed and correlation with pathological grade of response was measured.ResultsOrganoid models were treated with chemoradiation and both ST6GAL1 mRNA and protein significantly increased after treatment. The organoid model targeted with ST6GAL1 knockdown was found to have increased CC3 after treatment. In the tissue microarray, 42 percent of patient samples had an increase in percent tumor cell staining for ST6GAL1 after treatment. Post-treatment percent staining was associated with a worse grade of treatment response (p = 0.01) and increased staining post-treatment compared to pre-treatment was also associated with a worse response (p = 0.01).ConclusionST6GAL1 is associated with resistance to treatment in human rectal cancer and knockdown in an organoid model abrogated resistance to apoptosis caused by chemoradiation.

The ST6GAL1 sialyltransferase is overexpressed in multiple cancers including pancreatic ductal adenocarcinoma (PDAC). ST6GAL1 adds an α2-6-linked sialic acid to N-glycosylated membrane receptors, which consequently modulates receptor structure and function. While many studies have investigated the effects of ST6GAL1 on cell phenotype, there is a dearth of knowledge regarding mechanisms that regulate ST6GAL1 expression. In the current study, we evaluated the regulation of ST6GAL1 by two pro-inflammatory cytokines, IL-1β and IL-6, that are abundant within the PDAC tumor microenvironment. Cytokine activity was monitored using the Suit-2 PDAC cell line and two Suit-2-derived metastatic subclones, S2-013 and S2-LM7AA. For all three cell models, treatment with IL-1β or IL-6 increased the expression of ST6GAL1 protein and mRNA. Specifically, IL-1β and IL-6 induced expression of the ST6GAL1 YZ mRNA isoform, which is driven by the P3 promoter. The ST6GAL1 H and X isoforms were not detected. Promoter reporter assays confirmed that IL-1β and IL-6 activated transcription from the P3 promoter. We then examined downstream signaling mechanisms. IL-1β is known to signal through the NFκB transcription factor, whereas IL-6 signals through the STAT3 transcription factor. CUT&RUN experiments revealed that IL-1β promoted the binding of NFκB to the ST6GAL1 P3 promoter, and IL-6 induced the binding of STAT3 to the P3 promoter. Finally, we determined that inhibitors of NFκB and STAT3 blocked the upregulation of ST6GAL1 stimulated by IL-1β and IL-6, respectively. Together, these results highlight a novel molecular pathway by which cytokines within the tumor microenvironment stimulate the upregulation of ST6GAL1 in PDAC cells.

Efforts to develop targetable molecular bases for drug resistance for pancreatic ductal adenocarcinoma (PDAC) have been equivocally successful. Using RNA-seq and ingenuity pathway analysis we identified that the superpathway of cholesterol biosynthesis is upregulated in gemcitabine resistant (gemR) tumors using a unique PDAC PDX model with resistance to gemcitabine acquired in vivo. Analysis of additional in vitro and in vivo gemR PDAC models showed that HMG-CoA synthase 2 (HMGCS2), an enzyme involved in cholesterol biosynthesis and rate limiting in ketogenesis, is overexpressed in these models. Mechanistic data demonstrate the novel findings that HMGCS2 contributes to gemR and confers metastatic properties in PDAC models, and that HMGCS2 is BRD4 dependent. Further, BET inhibitor JQ1 decreases levels of HMGCS2, sensitizes PDAC cells to gemcitabine, and a combination of gemcitabine and JQ1 induced regressions of gemR tumors in vivo. Our data suggest that decreasing HMGCS2 may reverse gemR, and that HMGCS2 represents a useful therapeutic target for treating gemcitabine resistant PDAC.

PDF file - 104K, Supplemental Figure 1. Localization of CK2 in GBM Tumor and Mouse Brain, and in GBM Cells.

PDF file - 55K, Supplemental Figure 7. CX-4945 Abrogates GBM Cell Survival.

Aberrant glycosylation is a hallmark of a cancer cell. One prevalent alteration is an enrichment in α2,6-linked sialylation of N-glycosylated proteins, a modification directed by the ST6GAL1 sialyltransferase. ST6GAL1 is upregulated in many malignancies including ovarian cancer. Prior studies have shown that the addition of α2,6 sialic acid to the Epidermal Growth Factor Receptor (EGFR) activates this receptor, although the mechanism was largely unknown. To investigate the role of ST6GAL1 in EGFR activation, ST6GAL1 was overexpressed in the OV4 ovarian cancer line, which lacks endogenous ST6GAL1, or knocked down in the OVCAR-3 and OVCAR-5 ovarian cancer lines, which have robust ST6GAL1 expression. Cells with high expression of ST6GAL1 displayed increased activation of EGFR and its downstream signaling targets, AKT and NFκB. Using biochemical and microscopy approaches, including Total Internal Reflection Fluorescence (TIRF) microscopy, we determined that the α2,6 sialylation of EGFR promoted its dimerization and higher order oligomerization. Additionally, ST6GAL1 activity was found to modulate EGFR trafficking dynamics following EGF-induced receptor activation. Specifically, EGFR sialylation enhanced receptor recycling to the cell surface following activation while simultaneously inhibiting lysosomal degradation. 3D widefield deconvolution microscopy confirmed that in cells with high ST6GAL1 expression, EGFR exhibited greater co-localization with Rab11 recycling endosomes and reduced co-localization with LAMP1-positive lysosomes. Collectively, our findings highlight a novel mechanism by which α2,6 sialylation promotes EGFR signaling by facilitating receptor oligomerization and recycling.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies and is currently the third leading cause of cancer death. The aggressiveness of PDAC stems from late diagnosis, early metastasis, and poor efficacy of current chemotherapies. Thus, there is an urgent need for effective biomarkers for early detection of PDAC and development of new therapeutic strategies. It has long been known that cellular glycosylation is dysregulated in pancreatic cancer cells, however, tumor-associated glycans and their cognate glycosylating enzymes have received insufficient attention as potential clinical targets. Aberrant glycosylation affects a broad range of pathways that underpin tumor initiation, metastatic progression, and resistance to cancer treatment. One of the prevalent alterations in the cancer glycome is an enrichment in a select group of sialylated glycans including sialylated, branched N-glycans, sialyl Lewis antigens, and sialylated forms of truncated O-glycans such as the sialyl Tn antigen. These modifications affect the activity of numerous cell surface receptors, which collectively impart malignant characteristics typified by enhanced cell proliferation, migration, invasion and apoptosis-resistance. Additionally, sialic acids on tumor cells engage inhibitory Siglec receptors on immune cells to dampen anti-tumor immunity, further promoting cancer progression. The goal of this review is to summarize the predominant changes in sialylation occurring in pancreatic cancer, the biological functions of sialylated glycoproteins in cancer pathogenesis, and the emerging strategies for targeting sialoglycans and Siglec receptors in cancer therapeutics.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 β-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Increased cell surface sialylation is a prevalent feature of tumor cells. The addition of sialic acid (a negatively-charged sugar) to select surface receptors modulates the structure and function of these receptors, leading to changes in intracellular signaling and gene expression. Increased tumor cell sialylation occurs, in part, through the upregulation of sialyltransferases such as ST6GAL1, an enzyme that adds an α2-6 linked sialic acid to N-glycosylated proteins. ST6GAL1 is overexpressed in numerous malignancies, including pancreatic ductal adenocarcinoma (PDAC), and high expression correlates with a poor prognosis. Our group has shown that ST6GAL1 plays a causal role in promoting PDAC initiation and progression in tumor xenograft and genetically-engineered mouse (GEM) models. As one example, we developed a GEM model with conditional ST6GAL1 expression in the pancreas and crossed this line to the “KC” PDAC model, which expresses oncogenic Kras (KrasG12D). Mice with dual expression of ST6GAL1 and KrasG12D (”KSC” mice) exhibit greatly accelerated PDAC initiation, progression, and mortality when compared with KC mice. In light of ST6GAL1's known role in conferring progenitor-like cell characteristics, we postulated that ST6GAL1 activity contributes to PDAC initiation by fostering acinar to ductal metaplasia (ADM). During ADM, pancreatic acinar cells de-differentiate into ductal-like, progenitor cells and acquire greater proliferative potential as well as apoptosis resistance. Cells undergoing ADM are particularly vulnerable to Kras-dependent transformation. Using a variety of approaches and model systems including GEM organoids, 3D acinar cultures, acinar-like cell lines, and an in vivo assay for ADM, we uncovered a novel function for ST6GAL1 in promoting ADM. We further determined that high ST6GAL1 activity in acinar cells cooperates with KrasG12D to drive neoplasia. Finally, our studies suggest that the tumor-promoting effects of ST6GAL1 are mediated, at least in part, by ST6GAL1-mediated sialylation of EGFR. These collective results highlight a novel glycosylation-dependent mechanism involved in early stages of PDAC development. Citation Format: Susan L. Bellis, Asmi Chakraborty, Nikita Bhalerao, Michael Marciel, Jihye Hwang, Austin Silva. Role of ST6GAL1 sialyltransferase in early stages of pancreatic cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 336.

The ST6GAL1 sialyltransferase, which adds α2-6-linked sialic acids to N-glycosylated proteins, is upregulated in many malignancies including ovarian cancer. Through its activity in sialylating select surface receptors, ST6GAL1 modulates intracellular signaling to regulate tumor cell phenotype. ST6GAL1 has previously been shown to act as a survival factor that protects cancer cells from cytotoxic stressors such as hypoxia. In the present study, we investigated a role for ST6GAL1 in tumor cell metabolism. ST6GAL1 was overexpressed (OE) in OV4 ovarian cancer cells, which have low endogenous ST6GAL1, or knocked-down (KD) in ID8 ovarian cancer cells, which have high endogenous ST6GAL1. OV4 and ID8 cells with modulated ST6GAL1 expression were grown under normoxic or hypoxic conditions, and metabolism was assessed using Seahorse technology. Results showed that cells with high ST6GAL1 expression maintained a higher rate of oxidative metabolism than control cells following treatment with the hypoxia mimetic, desferrioxamine (DFO). This enrichment was not due to an increase in mitochondrial number. Glycolytic metabolism was also increased in OV4 and ID8 cells with high ST6GAL1 expression, and these cells displayed greater activity of the glycolytic enzymes, hexokinase and phosphofructokinase. Metabolism maps were generated from the combined Seahorse data, which suggested that ST6GAL1 functions to enhance the overall metabolism of tumor cells. Finally, we determined that OV4 and ID8 cells with high ST6GAL1 expression were more invasive under conditions of hypoxia. Collectively, these results highlight the importance of sialylation in regulating the metabolic phenotype of ovarian cancer cells.